It has been confirmed that Aspergillus filamentous fungi secrete a large amount of various enzyme proteins to the outside of the fungus body. For example, it is said that industrially used Aspergillus oryzae produces several tens grams or more of enzyme proteins per litter of culture medium. Thus, the use of filamentous fungi as a host enables secretion production of homogenous and heterogeneous proteins with high productivity. Furthermore, many strains of filamentous fungi have conventionally been used for production of brewing products, and so it can be said that the filamentous fungi are suitable for producing proteins also from the viewpoint of safety. Therefore, in recent years, examples in which filamentous fungi are used as a host for producing useful proteins by gene recombination has been reported. When producing secretory proteins, the amount of produced proteins is determined by various factors in the process from the expression of a targeted protein to the modification of the protein. The most effective means for increasing the production amount of proteins is to enhance the transcription efficiency, that is, to increase the amount of transcription. From such a viewpoint, promoters derived from various filamentous fungi have been isolated and protein production systems using the promoters have been reported to date. For example, a promoter of amylase gene of Aspergillus oryzae (see, for example, JP 62 (1987)-272988A and Biotechnology, 5, 368 (1987)), a promoter of glucoamylase gene of Aspergillus niger (see, for example, Biotechnology, 6, 1419 (1988)) have been isolated and used.
Under the present situation, although some promoters are used, little about a gene expression mechanism of the promoter is clarified. To date, only a factor involved in catabolite repression regulation as a transcriptional regulation factor (see, for example, Mol. Microbiol., 7, 847-857 (1993)), a binding factor of a HAP complex that is a wide-domain transcription activation factor (see, for example, Mol. Gen. Genet., 237, 251-260 (1993)), a transcription activation factor of starch degrading enzyme gene cluster (see, for example, Mol. Gen. Genet., 262, 668-676(1999)), and the like, have been reported. Therefore, examples of attempts that have been carried out to produce useful proteins using filamentous fungi with high efficiency include, mainly, producing bacteria with high productivity by classical breeding, and isolating naturally occurring higher expression promoter. However, such attempts have required much labor and been much dependent upon the contingency, so that possibility of obtaining high expression promoter is low. On the other hand, if the capability of expression control of promoters is improved, such problems could radically be resolved. Also to date, as an attempt to improve the capability of expression control, modification of α-glucosidase gene of Aspergillus oryzae has been reported (see, for example, JP 9(1999)-9968A and Appl. Microbiol. Biotechnol., 50, 459-467 (1998)). However, although with such modified promoter, the capability of expression control is improved to some extent, it cannot be said that such a sufficient expression property as to carry out the production of proteins can be obtained. There remains a demand for development of a promoter capable of transcribing the targeted protein gene with high efficiency.
The present invention was completed based on the above-mentioned background and has an object to provide a base sequence involved in expression regulation by a promoter. Furthermore, it is also an object of the present invention to provide a modified promoter with high expression activity by modifying a promoter based on the information of this base sequence. Furthermore, it is also an object of the present invention to construct an expression system (production system) of proteins using the filamentous fungus as a host by using this modified promoter.